Tuberculosis (TB) is a common infectious disease caused by mycobacterium, mainly M. tuberculosis. It usually attacks the lungs (as pulmonary TB) but can also affect the central nervous system, the lymphatic system, the circulatory system, the genitourinary system, the gastrointestinal system, bones, joints, and even the skin. TB is easily spread by airborne transmission of small droplets. Over 90% of the annual new TB cases and deaths occur in resource-poor and developing countries.
Sputum smear microscopy is the standard diagnostic method used for diagnosing TB. This method is more than 100 years old and fails to detect more than half of all active cases (Lalvani et al., Br. Med. Bull. 93, 69-84 (2010)). The interpretation of the tuberculin skin test relies on individual epidemiological risk factors of infection. This test therefore tends to be inaccurate under certain biological conditions and is incapable of distinguishing between latent TB and active TB infection. Additionally, it is labor-intensive for the patient as well as the health-care provider. Another diagnostic test for TB includes acid-fast bacilli staining of sputum sample. However, this test does not distinguish M. tuberculosis from non-tuberculosis mycobacteria and it requires an additional positive smear culture after 8 weeks for definitive diagnosis of pulmonary TB.
In industrialized countries, where TB burden is relatively low, TB diagnosis relies on molecular assays and mycobacterial cultures. In these countries, early positive results can be obtained using assays that determine the production of carbon dioxide or the consumption of oxygen by microorganisms in culture. However, approximately 20 days are required to obtain an accurate positive result.
Nucleic acid amplification tests (NAATs) that identify M. tuberculosis in respiratory system within 2-7 hours and interferon γ release assay are two of the more recently developed methods for TB diagnosis. These methods are considered to be more rapid, accurate and sensitive. However, the equipment used in these methods is expensive and it requires technical expertise and/or the testing requires the use of radioactive materials and their disposals. Hence, the current diagnostic techniques are either inaccurate and time consuming, or are expensive and demand highly sophisticated laboratories which are not available in resource-poor and developing countries.
Because of their potential role in the diagnosis of pulmonary diseases, exhaled Volatile Organic Compounds (VOCs) have captured an increased interest in recent years. Chemical analysis, including specific identification and quantification of VOCs in breath samples, has revealed key differences between breath compositions of patients afflicted with several pulmonary diseases as compared to control samples. These pulmonary diseases include asthma, chronic obstructive pulmonary disease, cystic fibroses and lung cancer (WO 2010/079491; U.S. 2013/143247; and U.S. 2013/150261).
The diagnosis of infectious diseases by detecting VOCs that are emitted from infected cells and/or the surrounding microenvironment has been performed. The diagnosis is based on the following principles of cell biology. The cell membrane of bacteria consists primarily of amphipathic phospholipids, carbohydrates and many integral membrane proteins that are distinct for different cell types. When a disease progresses, the cell undergoes structural changes that often lead to oxidative stress, i.e. a peroxidation of the cell membrane that induces VOCs emission. It has been shown that each type of infectious disease is characterized by a unique composition of VOCs (Dummer et al., Trends Anal. Chem., 30, 960-967 (2011)). These VOCs can be detected from samples of bodily fluids or the headspace of a container containing infected cells and/or tissues or directly from exhaled breath in which disease-related changes are reflected through exchange via the blood or directly via the lung airways (Zhu et al., J. Clic. Microbiol., 48, 4426-4431 (2010); and Naraghi et al., Sens. Actuat. B, 146, 521-526 (2010); Abaffy et al., PLOS ONE, 5(11), e13813, doi:10.1371/journal.pone.0013813 (2010); Sahgal et al., Br. J. Dermatol., 155, 1209-1216 (2006); Turner, Exp. Rev. Mol. Diag., 11, 497-503 (2011); and Pennazza et al., Sens. Actuat. B, 154, 288-294 (2011)).
Specific alterations in VOC compositions of urine and breath samples of TB positive individuals have been reported (Banday et al., Anal. Chem., 83(14), 5526-5534 (2011)). Phillips et al. used gas-chromatography linked with mass spectrometry (GC-MS) to identify TB-related VOCs, part of which was found in M. tuberculosis cultures (Tuberculosis, 90, 145-151 (2010); and Tuberculosis, 87, 44-52 (2007)). Analyzing these VOCs using pattern recognition algorithms provided the accurate classification of 80-84% of the samples (Phillips et al., Tuberculosis, 92, 314-320 (2012)). Banday et al. showed significant alterations in VOCs concentrations in urine samples collected from TB patients using GC-MS (Anal. Chem., 83, 5526-5534 (2011)).
The use of GC-MS analysis for the detection of VOCs as breath biomarkers for TB has several disadvantages for use in clinical point-of-care applications. In particular, this technique utilizes bulky equipment which is relatively expensive and complicated to operate. In addition, it involves a pre-concentrating step which increases the risk of contamination and/or loss of analytes (Buszewski et al., Biomed. Chromatogr., 21, 553-566 (2007)). Furthermore, the accuracy obtained in these measurements is relatively low and it does not meet the criteria which is required for a TB screening test. Syhre et al. used GC-MS for specific detection of nicotinic acid as an indication for active TB (Tuberculosis, 89, 263-266 (2009)). The method required the in-vitro methylation of the nicotinic acid prior to measurements. Furthermore, this method could not provide reliable results for smoking individuals. Peled et al. identified unique VOCs or a VOC profile in the breath of cattle infected with M. bovis (bovine tuberculosis) using GC-MS analysis. The unique profile of VOCs was used to design a nanotechnology-based array of sensors for detection of M. bovis-infected cattle via breath (Sens. Actuat. B, 171-172, 588-594 (2012); FIG. 11).
U.S. 2010/0291617 and U.S. 2009/0239252 disclose methods and devices for identifying M. tuberculosis bacteria in a sample comprising the detection of one or more volatile organic compounds indicative of a presence of or response to treatment or resistance of the M. tuberculosis bacteria in the sample.
U.S. 2010/0137733 discloses a method for detecting whether a subject has tuberculosis or monitoring a tuberculosis subject, said method comprising: contacting breath from said subject with an apparatus, said apparatus having a gas chromatograph, wherein said gas chromatograph is fluidly coupled to a detector array to produce a signal; and analyzing said signal from the detector array to determine whether said subject has tuberculosis.
U.S. 2007/0062255 discloses an apparatus for collecting and detecting compounds in a human breath sample comprising: a handheld sample collector comprising a sorbent phase; a breath analyzer comprising a thermal desorption column; two or more sensors for detection of breath compounds; and a flow controller for controlling the transfer of breath compounds from the sample collector into the breath analyzer, wherein the handheld sample collector and breath analyzer are configured for fluid communication with each other so that breath compounds from the sample collector can pass into the breath analyzer for detection.
U.S. 2004/0127808 discloses a method for assessing a disease in a subject, said method comprising: collecting condensate from a subject's breath, said condensate having an acetic acid or acetate concentration or both an acetic acid and acetate concentration; testing said condensate to determine said acetic acid or acetate concentration or both said acetic acid and acetate concentrations; and evaluating said acetic acid or acetate concentration or both said acetic acid and acetate concentrations to determine the presence, absence or status of a disease in the subject.
At present, no simple and reliable technique is available for TB diagnosis, suitable for population screening. There remains an unmet need for a rapid, accurate, and cost-effective TB diagnostic tool that can be used at resource-poor health facilities, including central reference laboratories, hospital and clinic laboratories, and ultimately at the point-of-care.